Sero- and Feco-Prevalence of Helicobacter pylori Infection and Its Associated Risk Factors among Adult Dyspeptic Patients Visiting the Outpatient Department of Adet Primary Hospital, Yilmana Densa District, Northwest Ethiopia

Background Most gastric cancers in the world are attributed to Helicobacter pylori (H. pylori) infections. The prevalence of H. pylori infection is influenced by socioeconomic status, hygiene, and lifestyle of the population. This study aimed to assess sero- and feco-prevalence of H. pylori infections and its associated risk factors among adult dyspeptic patients visiting the outpatient department of Adet Primary Hospital, Yilmana Densa District, northwest Ethiopia. Methods A hospital-based cross-sectional study was conducted from February 10, 2022 to April 10, 2022. The study participants were selected using a systematic random sampling technique. The data were collected by pretested questionnaires. Blood and stool specimens were collected from each patient for antibody and H. pylori antigen tests, respectively. The data were analyzed using Statistical Package for Social Science (SPSS) version 26.0. A logistic regression model was used to assess the strength of association between H. pylori positivity and risk factors. Results The sero- and feco-prevalence of H. pylori infection was 62% and 51.1%, respectively. In those patients who had a positive result with either the antibody (Ab) or antigen (Ag) test, rural residence (AOR = 5.55; 95% CI: 2.34–13.14; p < 0.001), alcohol consumption (AOR = 12.34; 95% CI: 2.29–66.51; p=0.003), having no awareness about H. pylori transmission (AOR = 4.76; 95% CI: 1.86–12.15; p=0.001), using pond/river as a source of drinking water (AOR = 5.22; 95% CI: 1.91–14.27; p=0.001), and open field defecation (AOR = 4.19; 95% CI: 1.67–10.52; p=0.002) were the explanatory risk factors significantly associated with H. pylori infection. Conclusion This study showed that the prevalence of H. pylori infection was very high. Most cases of gastric cancers are ascribed to H. pylori infection. Therefore, education should be given to communities so as to increase their awareness of the infection and the associated major explanatory risk factors identified in the present study.


Introduction
Helicobacter pylori (H. pylori) is a Gram-negative, microaerophilic rod-shaped bacterium with two to six lophotrichous fagella [1]. Numerous elements have been implicated as contributing factors for H. pylori's colonization and survival in the host's hostile stomach environment. environment of the gastric mucosa by hydrolyzing urea to ammonia and carbon dioxide [2,3].
Helicobacter pylori is an important pathogen, which is linked to a heavy burden of both malignant and nonmalignant disorders. Te bacterium is classifed as a human carcinogen and is highly linked to stomach cancer, which is the third most prevalent cancer-related death worldwide. It is also linked to disorders, including dyspepsia and peptic ulcer [4].
Tere are noticeable disparities in the epidemiology of H. pylori infection, particularly in children, between poor and developed countries [5]. Helicobacter pylori continues to be a signifcant global health issue, contributing signifcantly to morbidity and mortality from stomach cancer and peptic ulcer disease. Although it is estimated that half of the world's population has H. pylori infection, the prevalence of infection varies greatly between and within nations. Rapid economic development in developed nations, which boosted social standards, has been associated with a decline in infection rates. Despite the descending trend in prevalence worldwide, the overall prevalence in Africa remains very high (70%) [6]. However, these rates are more pronounced in children than in younger adults [7].
A previous systematic review and meta-analysis found out signifcant regional heterogeneity of H. pylori infections worldwide. It was shown that Oceania had the lowest prevalence (24.4%) compared to Africa which had the highest pooled prevalence (70.1%) of the infection [8]. A similar study on H. pylori infections further showed that Africa having the greatest global frequency of H. pylori infection [9].
In Ethiopia, the epidemiology of H. pylori exhibits striking heterogeneity across regions, study population, environmental factors, and lifestyles. In a meta-analysis study, the overall pooled prevalence of H. pylori infection was found to be 52.2%, with the Somali region having the highest prevalence (71%), followed by the Amhara region with a prevalence of 54.6% [10]. Te prevalence of H. pylori varied across geographical locations in Ethiopia; it was 42.8% in Mizan Aman town, Southwest Ethiopia [11], 72% among gastritis patients attending Yekatit 12 Teaching Hospital of Addis Ababa [12], 47.7% in Bekoji [13], 30.3% in Shashamane, Ethiopia [14], and 65.7% in Ziway, central Ethiopia (65.7%) [15]. Te frequency of H. pylori infection is also very high in the Amhara Regional State, but it is varied in diferent locations in the region. For example, the overall magnitude of H. pylori infection among dyspeptic patients attending Dessie Referral Hospital was 30.4% [16], 70.25% in Mekane Selam [17], and 34% in Debre Tabor [18].
Varied socioeconomic and hygienic conditions have been signifcantly associated with diferent prevalence rates of H. pylori infection, with individuals living in developed urban areas (less than 40%) being less infected than those living in rural developing areas (>80%) [19]. Te population's sociodemographics, socioeconomic position, personal cleanliness, and way of life all have an impact on the prevalence of H. pylori. Some of the risk factors for H. pylori infection include crowding, unsanitary environments, alcohol use, the type of toilet facility, and unreliable water supplies [20,21].
Serology tests are usually benefcial for seroepidemiological studies, but not for individual patient decision-making. In addition, they need to be confrmed for particular sites and evaluated for erroneous results emanating from cross-reactivity of antigens from various pathogens. Te accuracy of these tests might not go above 50% in areas with moderate H. pylori prevalence. However, the H. pylori stool antigen (HpSA) test is a more accurate way to identify recent and current H. pylori infection. In a single noninvasive test, the HpSA test has the ability to simultaneously test for antimicrobial resistance and give a speedy diagnosis [7].
According to Adet Primary Hospital's annual report, dyspepsia was ranked frst from the top 10 diseases in the year 2021. Even though several studies have been conducted in diferent parts of Ethiopia, there has not been any study about sero-and feco-prevalence and associated risk factors of H. pylori infection among adult dyspeptic patients visiting Adet Primary Hospital, Yilmana Densa District. Te research addressed the following research questions: what was the sero-and feco-prevalence of H. pylori infection among patients visiting Adet rimary Hospital? What were the major risk factors of H. pylori infection in the study area? What was the degree of association between the prevalence of H. pylori and potential risk factors? Te specifc objective of the present study was to determine the sero-and fecoprevalence, to identify the major explanatory risk factors, and also to determine the degree of association between the prevalence and the potential risk factors of H. pylori infection.

Study Area, Study Population, and Study Design.
Yilmana Densa District is located at a distance of 470 km from Addis Ababa (the capital city of Ethiopia) and 42 km away from Bahir Dar (the center city of the regional government). Te district is located at the geographical location of 11°29′ 60″ N latitude and 37°19′ 60″ E longitude ( Figure 1). Te topography of the district is 56% plain, 20% mountain, and 23% lowland (depression), and 1% is occupied by water bodies. Its elevation ranges from 1500 to 2523 meter above sea level. Te district is divided into the following three climatic zones: 64% Wayna Daga, 24% Dega, and 12% Kola. Te mean annual rainfall ranges from 822 mm to 2000 mm and the average range of temperature is 14°C-27°C. Te total population of the district in the year 2022, a projection based on the Ethiopian Census, was 228,725 of whom 111,120 were men and 117,605 were women. Of which, 26,198 or 11.5% were urban inhabitants and the rest 88.5% were rural inhabitants [22].
Tere are 11 health centers, fve private clinics, 40 health posts, and one primary hospital in the Woreda. Adet Primary Hospital is in the study area and serves 60,697 (male 33,345 and female 27,352) populations in the catchment area. A hospital-based cross-sectional study among adult dyspeptic patients visiting the Outpatient Department (OPD) of Adet Primary Hospital was conducted from February 10, 2022, to April 10, 2022.

Sample Size Determination.
Te sample size (n) was calculated using the single population formula for crosssectional surveys. Since there was no previous study conducted in the area, a 50% prevalence rate of H. pylori infection was taken, assuming that H. pylori is prevalent among patients in Adet Primary Hospital. Te sample size was calculated with a 95% confdence interval and a 5% margin of error [23] as follows: z is the critical value of the standard normal distribution, which is 1.96 at 95% CI, p is the estimated prevalence of H. pylori infection, and d (5%) is absolute precision. Based on the abovementioned formula, a sample size of 384 was obtained. To compensate for the nonrespondents and minimize errors probably arising from the likelihood of noncompliance, 5% was added. Terefore, the fnal sample size for the study involved 403 study participants.

Sampling Techniques.
Te study participants were selected using a systematic random sampling technique. On average, at Adet Primary Hospital, 20 adult dyspeptic patients attended the outpatient department each day. Terefore, in the 22 working days of the month, there were 440 dyspeptic patients, and for the two months, there were 880 dyspeptic patients who requested participation in the study. Te total expected number of dyspeptic patients during the study period was estimated to be 880. When the total population was divided by the sample size, the sample interval was two, and every two adult dyspeptic patients were selected until a total of 403 samples were obtained.
After obtaining written consent from each participant, a systematic random sampling technique was employed. Terefore, to get the required fnal sample size, the study participants were invited to take part in the study proportionally based on the number of dyspeptic patients who visited the outpatient department (OPD) during the study period. Tus, k � N/n, where "k" is the sample interval, "N" is the study population, and "n" is the sample size of the study �>880/403 � 2, and then the data were collected for every two adult dyspeptic patients who visited the OPD during data collection priorities.

Eligibility Criteria
2.4.1. Inclusion Criteria. Tose volunteers who signed the informed consent (18 years old and above) and also those who did not receive any anti-H. pylori treatment within the last four weeks prior to sampling were included.

Exclusion Criteria.
Tose patients who did not sign the informed consent, who were less than 18 years old, and those patients treated with any antibiotics within the last four weeks of study enrollment were excluded from the study.    2.6. Data Collection and Processing. Before data collection, written informed consent was obtained from study participants. Te data were collected using a pretested, structured, and self-administered questionnaire by trained data collectors. Prior to data collection, the questionnaire was administered to 5% of participants attending another health center other than Adet Primary Hospital with upper gastrointestinal symptoms to assess the clarity and understandability of the question. For participants who could not read and write, interviews were employed based on the structured questionnaire. Te questionnaire was developed in the Amharic language (the local language). Ten, the responses were translated back into English. Te completed questionnaires were collected and checked for completeness. Te blood and stool specimens were collected from each patient for H. pylori antigen and antibody tests, respectively, and immediately taken to the laboratory for processing. Some portion of the collected blood was used to determine the ABO and Rh blood groups by slide agglutination test using monoclonal anti-A, anti-B, anti-AB, and anti-D (Rh) antibodies according to the manufacturer's recommendation.
Some portion of the collected blood was allowed to clot in a test tube and centrifuged at 3000 rotations per minute for 10 minutes, and the serum was then separated. Te separated serum was examined serologically for H. pylori immunoglobulin G (IgG) antibodies using immune chromatographic rapid test kits (dBest H. pylori test kit, Ameritech Diagnostic Reagent Co., Ltd., Tongxiang, Zhejiang, China), which are nationally approved and used for serological diagnosis of H. pylori infection. Tis test contains a membrane strip, which is precoated with H. pylori capture antigen on the test binding region. Te H. pylori antigen-colloid gold conjugate and serum sample move along the membrane chromatographically to the test region (T) and form a visible line as the antigen-antibodyantigen gold particle complex forms. Tis test device has a letter of T and C as "Test Line" and "Control Line" on the surface of the case. Both the test line and control line in the result window are not visible before applying any samples. Te control line is used for procedural control. Te control line should always appear if the test procedure is performed properly and the test reagents in the control line are working. Te test was performed following the manufacturer's instructions.
Study subjects with dyspepsia were provided with a clean, dry, leak-proof, disinfectant-free, and wide-mouthed plastic container to collect about 10 g of fresh stool specimens into the container for the H. pylori antigen test. Instructions were given on how to prevent contamination of the stool with water and urine. Immediately after collection of stool specimens, H. pylori antigens in stools were detected against the SD BIOLINE test strip that utilized a monoclonal anti-H. pylori antibody conjugate based on a lateral fow chromatographic immunoassay technique. Specimens were tested using the SD BIOLINE stool antigen test strip (Zhejiang Orient Gene Biotech Co, Ltd, China) with 94.9%-100% sensitivity and 95-100% specifcity, according to the manufacturer's instructions. Te stool sample was transferred to a vial with diluents, vigorously agitated, and after two minutes of resting the tube, dropped around two to three drops (80 mL) into the round window of the test cassette. Reading was performed after 10 minutes of incubation at room temperature, and based on the appearance of colored lines across the central window of the cassette, two lines, C (control) and T (test), indicate a positive test; only one line in C indicates a negative result. A pale-colored line in T is also considered positive.

Data
Analysis. Data were analyzed using SPSS version 26.0 (SPSS Inc, Chicago, IL, USA). Descriptive statistics were used to characterize data and assess the distribution of study variables. Categorical variables were summarized in frequencies or percentages. A Chi-square test was performed to check for the presence of an association between dependent variables and independent variables. Univariate logistic regression was also carried out to identify and select variables with p < 0.25, which were candidates for the multivariate logistic regression model [24]. A multivariate logistic regression model was used after adjusting for confounding variables to identify the major explanatory risk factors for H. pylori infection among study participants. Variables with a p value <0.05 in the fnal model of multivariate logistic regression were considered statistically signifcant.

Ethical Considerations.
Before conducting the investigation, the investigator obtained ethical clearance (Ref. No., PRCSVD/291/2014) from the Ethical Clearance Committee of Science College, Bahir Dar University. A letter describing the objective of the research was written to Adet Primary Hospital. Ten, authorities at Adet Primary Hospital were informed and asked for permission. An informed, voluntary, written, and signed consent was obtained from the study subjects after explaining the importance of the study briefy. Participation in the study was on a voluntary basis, and participants were informed about their full right to withdraw or refuse at any stage of the study if they did not want to participate. Moreover, the confdentiality of the study participants was also maintained. Dyspeptic patients were tested for H. pylori following the national standard diagnosis guideline. Finally, the study participants whose test results were positive were treated with standard drugs as prescribed by a physician from Adet Primary Hospital.

Sociodemographic and Socioeconomic Characteristics of Study Participants.
A total of 403 study subjects participated in this study. Te response rate was 100%. Among them, 210 (52.1%) were males and 193 (47.9%) were females. Te age of the participants ranged from 18 to 64 years, with a mean and standard deviation of 38 ± 11.2 years. Te majority (264; 65.5%) of the study participants were rural dwellers. More than half of the study participants (250; 62%) were illiterate. Two hundred twenty-nine (56.8%) of the study participants were married and 68 (16.9%) were single. More than half of the study participants (214; 53.1%) were farmers and 16 (4%) were students. Of the total, 116 (28.8%), 200 (49.6%), and 87 (21.6%) came from a family size of >5, 4-5, and ≤3, respectively. Two hundred thirty-two (57.6%) of the study participants had a monthly income of 1000-2500 Ethiopian Birr (ETB) and 88 (21.8%) had a monthly income below 1000 ETB (Table 1).  Table 3). Te 95% confdence interval for prevalence estimate is given as supplementary document (Table S1).

Sero-and Feco-Prevalence of H. pylori Infection.
Out of the 403 examined adult dyspeptic patients, the number of positive patients who were detected by the serological test was 250, i.e., a sero-prevalence of 62%. Te number of individuals who were found positive by serological test and became negative by stool antigen test was 55 (13.6%).
Out of the total examined patients, the number of positive patients detected by the stool antigen test was 206 (51.1%), i.e., the feco-prevalence was 51.1%. Te number of individuals who were found positive by stool antigen test and became negative by the serological test was 11 (2.7%). Te number of positive patients who were detected by both serological and stool antigen test was 195 (48.4%). From the total, the number of individuals who were positive for either Ab or Ag test was 261 (64.8%) (Figure 2).   In the fnal model of multivariate logistic regression, fve independent variables, i.e., residence, alcohol consumption, awareness about the transmission mode of H. pylori, source of drinking water, and type of toilet facility, remained signifcantly associated with H. pylori infection, and they were found to be signifcant explanatory risk factors (p < 0.05) for H. pylori infection among dyspeptic patients during the study period (Table 4).
In the fnal multivariate regression, it was found that males were 1.89 times at higher risk of H. pylori infection (AOR � 1.89; 95% CI: 0.86-4.15; p � 0.111) than females, even though, it was not statistically signifcant. Even though it was not a signifcant explanatory risk factor, those participants who were above 50 years old were 1.61 times at higher risk of acquiring H. pylori infection (AOR � 1.61; 95%
Te result of this study also showed that the odds of H. pylori infection were about 12.34 times higher among dyspeptic patients who consumed alcohol than those who did not consume alcohol. Tis fnding is in agreement with studies conducted in Uganda (21), Bekoji (southeast of Ethiopia) [13], Cameron [61], Shashamane (Ethiopia) [14], Dessie (Ethiopia) [16], Indonesia [62], and Hosaena (Ethiopia) [47]. It is highly likely that alcohol can directly damage the gastric mucosal layer, favouring the attachment of the bacterium to the wall of the stomach. Moreover, frequent and heavy drinkers can possibly predispose them to social contacts that favor transmission of H. pylori infection.
Tose adult dyspeptic patients who did not have awareness about the transmission of H. pylori infection were 4.76 times more likely to be at higher risk of acquiring H. pylori infection than those who had awareness. Tis might be due to the fact that those who do not have awareness of the transmission of H. pylori infection may not take appropriate preventive measures to protect themselves from the infection. Lack of awareness means there has been no action taken to avoid the infection. However, in contrast to the fnding of the current study, a previous study conducted on the prevalence of H. pylori infection and its associated risk factors among patients undergoing upper gastrointestinal diagnosis in Shashamane Referral Hospital in Shashamane, Ethiopia, reported that awareness about H. pylori infection was not a signifcant risk factor associated with H. pylori infection [14].
Likewise, the multivariate analysis of this study also showed that the odds of H. pylori infection were about 5.22 times higher among dyspeptic patients who used a pond/river as a source of drinking water. Most people in the study area who did not have toilets might have defecated in the open feld, which could contaminate water bodies used for drinking and food preparation, thereby increasing the chance of acquiring an H. pylori infection. Tis fnding is in line with that of studies conducted in Uganda [63], Mizan Aman (Ethiopia) [11], Mekane Selam (Ethiopia) [17], Gondar University Hospital (Ethiopia) [45], Cameroon [61], Sekota (Ethiopia) [52], United Arab Emirates [56], Egypt [42], Pakistan [64], Alaska, U.S.A. [46], Nigeria [43], Debre Tabor (Ethiopia) [18], and Yemen [65].
Study participants who practiced open feld defecation were 4.19 times at higher risk for H. pylori infection than those who used an improved latrine. Tese higher odds of infection might be associated with not having a toilet, leading them to defecate in the open feld and at the same time exposing themselves and others for the acquisition of the bacterium from the open feld. Tis fnding is in agreement with studies conducted in Mizan Aman (Ethiopia) [11], Debre Tabor (Ethiopia) [18], and Ziway (Ethiopia) [15]. In contrast to the fnding of the present study, the type of toilet used, however, was not signifcantly associated with H. pylori infection in studies conducted at Jasmine Internal Medicine and Pediatrics Specialized Private Clinic in Addis Ababa city (Ethiopia) [66].
Generally, the fndings of the present study could provide information that can be utilized in the planning of meaningful public health control programs in the prevention and control of H. pylori infection. Furthermore, it also provides information on the major explanatory risk factors of H. pylori infection in the study area.

Limitation of the Study
Tis study was limited only to adult dyspeptic patients attending the outpatient department of the Adet Primary Hospital from February 10 to April 10, 2022. Te study did not include adult dyspeptic patients visiting other health centers in the district. Te employment of a rapid serology (IgG) test kit for the diagnosis could not detect active H. pylori infection because high-titer IgG can last for months to years. Tis may overestimate the prevalence of H. pylori infection among the study participants since the total positivity was determined by the combination of the two tests. Moreover, this study was limited only to symptomatic patients visiting the hospital. Terefore, it might not show the actual prevalence of the infection in the entire population of the district.

Conclusion
Tis study revealed a high (64.4%) overall prevalence of H. pylori infection among adult dyspeptic patients attending the Adet Primary Hospital. Te sero-and feco-prevalence of H. pylori infection in this study were 62% and 51.1%, respectively. Rural residence, lack of awareness on H. pylori transmission, alcohol consumption, using a pond/river as a source of drinking water, and open feld defecation were found to be signifcant explanatory risk factors associated with H. pylori infection among study participants. However, none of the clinical characteristics considered in the present study were signifcantly associated (p>0.05) with H. pylori infection among the studied subjects.
Te present fndings have potential implications for clinical practice and public health in the study area. Te very high prevalence of H. pylori infection among the studied population calls for certain intervention mechanisms (e.g., education) by concerned bodies, as most gastric cancer cases are associated with this particular infection. Although not statistically signifcant, close to 65% of study participants infected with H. pylori (Table 2) had a history of epigastric pain, heartburn, abdominal fullness, nausea, belching, melena, vomiting, and bloody vomiting. Tese clinical signs and symptoms can be used by clinicians to suspect H. pylori infection and accordingly order patients for the diagnosis of H. pylori active infection from stool specimens. At the community level, H. pylori infection and its efects (e.g., gastric cancer) and associated factors such as consumption of alcohol, source of drinking water, and type of toilet facility should be further studied to reduce the prevalence and transmission of H. pylori.

Data Availability
Te data that support the fndings of the study are available within the article and raw data can be obtained from the corresponding author upon request.

Disclosure
Te study was performed as part of the employment of Bahir Dar University, Bahir Dar, Ethiopia. Bahir Dar University was not involved in manuscript writing, editing, approval, or decision to publish. Tis research was previously presented as a thesis [68].

Conflicts of Interest
Te authors declare that they have no conficts of interest.